Development of vaginal microbicides to safely prevent sexual transmission of herpes simplex virus (HSV), a[unreadable] major co-factor for HIV acquisition, is a major health priority. We seek to determine whether RNA[unreadable] interference (RNAi) can be harnessed to prevent transmission and acquisition of HSV and provide a novel[unreadable] strategy for microbicide development. We hypothesize that short interfering double-or single-stranded RNA[unreadable] (siRNA) can be specifically targeted to the vaginal mucosa to decrease expression of cellular and/or viral[unreadable] proteins critical for the establishment of HSV infection. The first priority will be to target cellular proteins[unreadable] required for viral entry. We hypothesize that gene silencing of either the HSV co-receptor, nectin-1, which[unreadable] has been demonstrated to be the major co-receptor for HSV infection of vaginal tissue, and/or focal adhesion[unreadable] kinase (FAK), activation of which plays a pivotal role in entry and nuclear transport of HSV capsids, will[unreadable] inhibit HSV infection. In parallel studies, we will also target candidate viral genes that encode for proteins[unreadable] essential for viral replication, cell-cell spread and immunomodulatory genes. The choice of viral targets will[unreadable] build from experiences gained from studies of HSV deletion viruses that have been evaluated for gene[unreadable] therapy or as vaccine candidates. Enthusiasm for this approach derives from preliminary results obtained for[unreadable] HSV and HIV. A single mucosal application of liposomal complexes of siRNA targeting nectin-1 (HSV) or[unreadable] CCR5 (HIV) decreases gene expression. Transfection of human cervical cells with siRNA targeting FAK[unreadable] substantially inhibits HSV infection. We propose to translate these findings to mucosal models of genital[unreadable] herpes. This NIH Multi-project Cooperative Agreement will apply an iterative approach toward identifying the[unreadable] best siRNA target(s), optimal modification of siRNA, and formulations to prevent genital herpes. In parallel[unreadable] studies, the mucosal response to siRNA application will be evaluated in human cell cultures and in a murine[unreadable] model as it is critical that topical therapy not interfere with innate genital tract defenses. The results obtained[unreadable] may provide compelling support for advancing siRNAs to clinical trials.